Composition and method for hip1-targeting inhibitor compounds

ABSTRACT

Provided herein is an inhibitor compound targeting Hip1, with the inhibitor compound comprising a tripeptide targeting sequence that directs the compound to the active site of Hip1 and a C-terminal electrophilic warhead conjugated to the targeting sequence, the warhead configured to inactive the enzyme.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/217,172 entitled “COMPOSITION AND METHOD FOR HIP1-TARGETINGINHIBITOR COMPOUNDS” and filed on Jun. 30,2121 for Nathan E. Goldfarb,which is incorporated herein by reference.

COLORED DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIELD OF THE INVENTION

This invention relates to molecular targeting and more particularlyrelates to Hip1 inhibitor targeting drugs including for tuberculosistreatment.

BACKGROUND

According to the World Health Organization, in 2018, 1.5 million peopledied from Mycobacterium tuberculosis (Mtb) (including 251,000 peoplewith HIV). New drugs for the treatment of Tuberculosis (TB) are direlyneeded. This is due to the evolution of drug resistant strains of Mtb,the causative agent of TB. Also, many of the current FDA approved drugsused to treat drug resistant strains of TB are injectables, have toxicside effects, and require a six month treatment regimen. Therefore, aneed exists for a therapeutic compound narrowly targeting Mtb for aneffective and rapid course of treatment. Beneficially, such a compoundwould be free of toxic side effects.

SUMMARY OF THE INVENTION

The foregoing discussion illustrates that a need exists for aneffective, short-term treatment for Mtb, the treatment being free oftoxic side effects. The present invention has been developed in responseto the present state of the art, and in particular, in response to theproblems and needs in the art that have not yet been fully solved bycurrently available Mtb treatments. Accordingly, the present inventionhas been developed to provide a class of inhibitors of Hydrolaseimportant for pathogenesis (Hip1), a drug target that overcomes many orall of the above-discussed shortcomings in the art.

Reference throughout this specification to features or similar languagedoes not imply that all of the features that may be realized with thepresent invention should be or are in any single embodiment of theinvention. Rather, language referring to the features is understood tomean that a specific feature or characteristic described in connectionwith an embodiment is included in at least one embodiment of the presentinvention. Thus, discussion of the features and characteristics, andsimilar language, throughout this specification may, but does notnecessarily, refer to the same embodiment.

Furthermore, the described features, and characteristics of theinvention may be combined in any suitable manner in one or moreembodiments. One skilled in the relevant art will recognize that theinvention can be practiced without one or more of the specific featuresor characteristics of a particular embodiment. In other instances,additional features and characteristics may be recognized in certainembodiments that may not be present in all embodiments of the invention.These features and characteristics of the present invention will becomemore fully apparent from the following description and appended claimsor may be learned by the practice of the invention as set forthhereinafter.

Provided herein is a novel class of potent inhibitors of Hip1, a novelcocrystal structure of a potent Hip1-directed inhibitor bound to Hip1,and a novel chromogenic substrate for Hip1. Hip1 is an Mtb serinehydrolase/protease that has emerged as a promising drug target for thedevelopment of novel TB drugs. Hip1 is an Mtb cell wall-associatedserine hydrolase that plays an important role in the pathogenicstrategies of Mtb cell envelope maintenance and the dampening of hostcell proinflammatory responses. Functional studies identify Hip1 as atarget for drug discovery. Mice infected with a Hip1 transposon mutantstrain survive significantly longer than wild-type Mtb-infected mice andexhibit mild lung immunopathology despite high bacterial burdens (1,2).

The illustrated embodiment of the class of Hip1 inhibitor compoundsincluding NS-049-2, a low molecular weight (696.72 g/mole), potentinhibitor (K_(i)=309±15 pM) of its drug target, Hip1. To date, no otherdrug-like, tight binding inhibitors target Hip1. As a result, antibioticresistance has not evolved against Hip1-directed therapeutics.

Further provided herein is a class of compounds for the inhibition ofHip1, the compounds comprising a tripeptide targeting sequence,Phe-Lys-Leu, that directs the compound to the active site of Hip1; and aC-terminal alpha-keto methyl ester electrophilic warhead conjugated tothe targeting sequence, the warhead configured to inactivate the enzyme.In some embodiments the compound comprises:

Also provided are position 1 (P1) derivatives of the compound ofcomprising Cbz-Phe-Lys-Gln-COCO₂Me, Cbz-Phe-Lys-Gln lactam-COCO₂Me,Cbz-Phe-Lys-Asn-COCO₂Me, Cbz-Phe-Lys-Glu-COCO₂Me,Cbz-Phe-Lys-Val-COCO₂Me, and Cbz-Phe-Lys-(X)-COCO₂Me, where (X)=anyamino acid, amino acid derivative, or chemistry.

Further provided here are P3 derivatives of the compound comprisingCbz-Tyr-Lys-Leu-COCO₂Me , Cbz-Nle-Lys-Leu-COCO₂Me, andCbz-(X)-Lys-Leu-COCO₂Me; where (X)=any amino acid, amino acidderivative, or chemistry.

In certain embodiments P1 and P3 derivatives compriseCbz-(X_(n+1))-Lys-(X)-COCO₂Me, where (X_(n+1)) and (X)=any amino acid(s), amino acid derivative (s), or chemistry (ies). TruncatedDerivatives may comprise one or more of Cbz-Leu-COCO₂Me, Cbz-X-COCO₂Me ,where (X)=any amino acid, amino acid derivative, or chemistry,Cbz-Lys-Leu-COCO₂Me, and Cbz-Lys-X-COCO₂Me, where (X)=any amino acid,amino acid derivative, or chemistry.

N-terminal Lengthened Derivatives as provided herein may compriseCbz-(X)_(n+1)-Phe-Lys-Leu-COCO₂Me; (where (X)_(n+1=)any amino acid(s),amino acid derivatives, or chemistries.

Also provided herein are Protecting Group Derivatives comprising(Z)-(X)_(n+1)-(X)-Lys-(X)-COCO₂Me, where (Z)=any protecting group,(X)_(n+1=)any amino acid(s), amino acid derivatives, and (X)=any aminoacids, amino acid derivatives, or chemistries. In certain embodimentsProtecting Group Derivatives comprise Cbz-Phe-Lys-Leu-pNa(pNa=paranitroanilide) or derivatives of this compound includingconjugated to various chromophores, fluorophores, antibodies,nanobodies, or other reporter groups for the design of novel enzymaticactivity assays, serine protease purification methods, molecular probesfor the detection of novel serine/threonine proteases, and rapiddiagnostic tests for the presence of Mtb in patients.

Also provided herein are Electrophilic Warhead Derivatives comprising(Z)-(X)_(n+1)-(X)-Lys-(X)-(Y), where (Y)=any electrophilic group capableof forming a covalent attachment to active site Ser228 of Hip1, where(Z)=any protecting group, (X)_(n+1=)any amino acid(s), amino acidderivatives, and (X)=any amino acids, amino acid derivatives, orchemistries.

Additionally provided herein are methods for purification of Hip1 andthe methods for cocrystallization Hip1 bound with NS-049-2, as well asthe X-ray cocrystal structure of Hip1 bound with NS-049-2.

The compounds and derivatives herein provided may be used to treat atleast tuberculosis, as molecular probes for the detection ofserine/threonine proteases involved in pathophysiological processes, asa diagnostic assay for the detection of Mtb including from patientsputum samples and as a booster to the BCG vaccine.

In some embodiments the compounds herein are disclosed as a booster tothe BCG vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of the inhibitor compound as determined byNMR;

FIG. 2A shows that the inhibitor kills Mycobacterium tuberculosis inliquid culture in a direct killing assay with an MIC=1.32±1.4 μM;

FIG. 2B shows that the inhibitor inhibits intracellular growth ofMycobacterium tuberculosis in its host cell, the macrophage, with anIC₅₀=6.3±1.1 μM;

FIG. 2C shows that the inhibitor exhibits minimal cytotoxicity with RAWmacrophages;

FIG. 2D shows that the inhibitor exhibits minimal cytotoxicity withHepG2 hepatocytes;

FIG. 3A is the 2.7 Å X-Ray cocrystal structure of the inhibitor compound(green sticks) herein covalently bound in the active site of Hip1;

FIG. 3B shows well defined electron density (light blue chicken-wire)for the inhibitor (green sticks) covalently bound to the active siteserine 228 of Hip1;

FIG. 3C shows van der Waals interactions (yellow dashes) that stabilizethe inhibitor in the active site cleft of Hip1. The sidechains of theinhibitor fit into their respective pockets, labeled S1-S3, in theactive site.

FIG. 3D shows polar interactions (black dashes) that stabilize theinhibitor (green sticks) in the active site cleft of Hip1. Watermolecules are rendered as red spheres.

FIG. 4 shows the line structure for the novel Hip1 chromogenic substratedetermined by NMR.

DETAILED DESCRIPTION OF THE INVENTION

Reference throughout this specification to “one embodiment,” “anembodiment,” or similar language means that a particular feature,structure, or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” andsimilar language throughout this specification may, but do notnecessarily, all refer to the same embodiment.

Furthermore, the described features, structures, or characteristics ofthe invention may be combined in any suitable manner in one or moreembodiments. In the following description, numerous specific details areprovided. One skilled in the relevant art will recognize, however, thatthe invention can be practiced without one or more of the specificdetails, or with other methods, components, materials, and so forth. Inother instances, structures, materials, or operations that are known inthe art are not shown or described in detail to avoid obscuring aspectsof the invention.

With a global market in the billions of dollars, FDA approved proteaseinhibitors have been very successful in treating a number of diseasesincluding the Human Immunodeficiency Virus, Type 2 diabetes, Hepatitis,and obesity, to name a few. One drug development strategy is to design acompound comprising a moiety that targets the compound to the proteasedrug target. Conjugated to the targeting moiety is a “warhead” thatinactivates the enzyme. An example of this approach is the FDA approvedprotease inhibitor, Bortezomib (Velcade®; Takeda) which is indicated forthe treatment of multiple myeloma and mantle cell lymphoma. It containsa peptidomimetic targeting sequence conjugated to a boronic acid“warhead” that inactivates the catalytic threonine residue of itstarget, the 26 S proteasome.

FIG. 1 is a schematic line drawing depicting the structure of theillustrated embodiment of the inhibitor compound provided herein. Theillustrated inhibitor compound is a potent inhibitor (K_(i)=309±15 pM)of Hip1. The inhibitor compound contains a tripeptide targetingsequence, Phe-Lys-Leu, that directs the compound to the active site ofHip1. A C-terminal alpha-keto methyl ester electrophilic warhead isconjugated to the targeting sequence. The warhead acts to quiesce theactivity of the active site serine 228 residue of the enzyme, thusrendering the drug target inactive.

Advantages of this compound include its potency for Hip1 (K_(i)=309±15pM), low molecular weight (696.72 g/mole), and its drug target, Hip1. Todate, no other existing drug-like, tight binding inhibitors target Hip1.As a result, antibiotic resistance has not currently evolved againstHip1-directed therapeutics. Additionally, inhibition of Hip1 may boostthe host's immune response to help clear the infection. No other anti-TBdrug on the market uses this strategy to promote bacterial clearance.Furthermore, since Hip1 plays an important role in Mtb cell envelopmaintenance, using NS-049-2 in conjunction with current FDA approvedantibiotics may result in greater bacterial clearance efficacy.According to a recent report an Mtb transposon mutant of Hip1 exhibitssevere growth attenuation in the presence of 0.5 μg/mL of ethambutol,which affected the growth of WT Mtb only marginally. This mutant alsoshowed increased sensitivity to the antibiotics meropenem, vancomycin,and rifampicin. This indicates that inhibition of Hip1 may result inreduced Mtb fitness at partial inhibitory antibiotic concentrations.Therefore, such Mtb treatment regimens may require lower concentrationsof antibiotics and/or a reduced treatment time, potentially increasingpatient compliance and reducing toxic side effects. It should be notedthat the use of multiple drugs (combination therapy) is a verysuccessful approach in treating a number of bacterial, viral, fungal,and parasitic infections.

FIG. 2A is a direct killing assay showing the efficacy of the inhibitoragainst Mtb grown in liquid culture. The inhibitor kills Mtb with anMIC=1.32±1.4 μM. MICs were performed as described in Ollinger et al.,2013.

FIG. 2B shows growth inhibition of Mtb when tested in an intracellularRAW macrophage assay. RAW macrophages were infected with Mtb overnightand then treated with the inhibitor at various concentrations for sevendays at which time the cytotoxicity of the inhibitor on Mtb wasassessed, as previously mentioned. The inhibitor has an IC₅₀=6.3±1.1 μM.

FIG. 2C shows RAW macrophages dosed with various concentrations of theinhibitor. Importantly, the compound shows minimal cytotoxicity towardsRAW macrophages, with a TC₅₀ value >100 μM.

FIGS. 2D shows plot of HepG2 hepatocytes versus the concentration of theinhibitor. Importantly, the compound shows minimal cytotoxicity towardsHepG2 hepatocytes, with a TC₅₀ value >100 μM.

FIGS. 3A to 3D illustrate the three-dimensional atomic X-ray cocrystalstructure of Hip1 bound with the compound herein. Inspection of thestructure reveals that the compound forms a covalent interaction withthe active site Ser228 of Hip1, thus rendering the enzyme inactive. Thisconstitutes a novel ligand binding mode in the active site of Hip1.

FIG. 3A illustrates the 2.7 Å cocrystal structure of Hip1 (blue ribbon)covalently bound with the inhibitor (green sticks). The amino acidresidues composing the catalytic triad of Hip1 are rendered as yellowsticks.

FIG. 3B illustrates the well-defined electron density (light bluechicken wire; σ level=1.0) for the inhibitor (green sticks) covalentlybound to the active site Ser228 (yellow sticks) of Hip1, thusinactivating the enzyme. The absence of electron density for the Cbzprotecting group suggests that it is disordered in the structure.

FIG. 3C illustrates the binding mode of the inhibitor (green sticks) inthe active site of Hip1. Active site residues that make extensive vander Waals (vdw) interactions (yellow dashes) are rendered as orangesticks. Nonpolar contacts within 4.2 Å were assigned as vdwinteractions. The inhibitor is stabilized in the active site throughextensive vdw interactions. The active site Ser228 is colored yellow.

FIG. 3D illustrates the polar interactions (black dashes) made betweenthe inhibitor (green sticks) and the active site residues of Hip1(yellow, orange and blue sticks) and water molecules (red spheres). Thesalt bridge between P2 Lys of the inhibitor and the carboxylate ofGlu113 explains the pronounced selectivity for Lys observed in substrateprofiling experiments (Lentz et al., 2016);

FIG. 4 illustrates the line structure of the substrate analogue derivedfrom the inhibitor (K_(m)=1.2±0.23 μM; data not shown). The novelchromogenic Hip1 substrate is useful for enzymatic characterization anddetection of Hip1. Upon Hip1-dependent cleavage of the peptide bondbetween P1 leucine and the para-nitroanalide group, there is an increasein absorbance at 405 nm as detected by a spectrophotometer orplate-reader.

The inhibitor compound herein exhibits high potency for Hip1(K_(i)=309±15 pM) and has the advantage of low molecular weight (696.72g/mole). Additionally, the compound herein may be a reversibleinhibitor. Pharmaceutical companies historically prefer to developreversible inhibitors rather than irreversible inhibitors, as reversibleinhibitors typically exhibit lower toxicity in the case of off-targetbinding.

This disclosure, and the supporting work represent the firstcrystallographic determination of a ligand binding mode in the activesite of Hip1. Such an atomic roadmap of the Hip1 active site opens theway to populate this novel class of Hip1 inhibitor compounds and torefine inhibitor compounds to diminish any pharmaceutical liabilities(structure-based refinement). The inhibitor compound class hereinrepresents a novel class of compounds that may be translated into a newdrug useful for the treatment of Tuberculosis.

Rapid diagnosis of active TB in patients with negative sputum smears foracid fast bacteria may enable prompt, highly accurate identification ofdrug-resistant strains of M. tuberculosis. The inhibitor compound classherein or its derivatives may be used to develop a diagnostic test forthe presence of Mtb in patient sputum samples. The compound herein orits derivatives represent scaffolds for the development of novelmolecular probes useful for the detection of Mtb in patients. Forexample, an ELISA assay may be developed predicated on an antibodyconjugated to the compound herein or its derivatives. If Mtb is presentin the patient's sputum, the inhibitor-antibody probe will bind to Hip1on the surface of Mtb. A secondary antibody is then added to generate adetectable signal. Variations of this assay may utilize other bindingsystems, for exampe the biotin-streptavidan system.

Rapid, field-ready assays may also benefit from the potency of thenovel, chromogenic substrate Cbz-Phe-Lys-Leu-paranitroanalide(K_(m)=1.2±0.23 μM) described herein. Upon Hip1-dependent cleavage ofthe Leu-pNa bond, there is an increase in absorbance at 405 nM. Patientsputum samples positive for TB will contain Hip1, thus will react withthe substrate to yeild a change in absorbance which can be read on aportable spectrophotometer. Nonspecific cleavage of the substrate can bemitigated by the inclusion of protease inhibitors that do not inihibitHip1 but do inhibit other proteases.

Finally, the inhibitor compound herein or its derivatives may be used asmolecular probes useful for the discovery of other protease drug targetsfor Tuberculosis, as well as probes for serine/threonine proteasesinvolved in the pathologies of other diseases. The inhibitor compoundherein may also be useful as a booster for the bacilli Calmette-Guerin(BCG) vaccine which is has variable efficacy (0-80%) in preventingpulmonary TB (3). Since Hip1 plays a role in suppression of the host'simmune response, the compound herein may serve to boost a patient'simmune response when given in concert with the BCG vaccine, thusincreasing the efficacy of the vaccine.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

EXAMPLES Example 1. Recombinant Expression and Purification of Hip1

An N-terminally truncated recombinant Hip1 was generated using PCRdeletion mutagenesis using a full length clone of Hip1 as template DNA.The truncated gene was subcloned into the glutathione S-transferase(GST) expression vector, pGEX-6P-1 (Amersham Biosciences).Overexpression of the Hip1-GST fusion protein was achieved in E. coliBL21 DE3 cells, cells lysed by sonication, and the fusion proteinharvested as insoluble inclusion bodies. Using a modified method ofWestling et al. the inclusion bodies were solubilized at 1 mg/mL in 8 Murea containing 0.05 M CAPS, 0.005 M EDTA and 0.18 Mbeta-mercaptoethanol with stirring at 25° C. for 45 minutes. To removeinsoluble material, the mixture was centrifuged at 17,500×g for 30 minat 25° C. The clarified material was refolded by dialysis against5.7×its volume of 0.05 M Tris-HC1, 0.005 M EDTA, 2 mM reducedglutathione, 0.4 mM oxidized glutathione, pH 7.3, at 4° C. withstirring. The dialysis buffer was replaced with fresh refolding bufferafter 2 hours and protein was let refold for another 2 days. Toconcentrate the Hip1-GST fusion protein, it was centrifuged 20-30 min,30,000×g, 4° C., and loaded onto 5×5 mL HiTrap Q FF columns equilibratedin 20 mM Tris-HCl, 10 mM NaCl, pH 8 (Buffer A). The column was washed at1 mL/min for 70 min with Buffer A followed by a gradient elution of10-100% Buffer B (20 mM Tris-HCl, 1.5 M NaCl, pH 8). To cleave theHip1-GST fusion protein, 330 Units PreScission Protease® (Genscript) wasadded to 33 mg fusion protein in 20 mM Tris, 250 mM NaCl, pH 7.5, 1 mMDTT, 1 mM EDTA, 1% Tween 20 in a final volume of 4.8 mL. The reactionwas rotated overnight at 4° C. N-terminal sequencing of recombinant Hip1following PreScission Protease® cleavage indicates that the PreScissionProtease® cleaves the fusion protein at the correct processing siteyielding the expected N-terminus of Hip1: NH2-GPLG. To separate Hip1from GST, the PreScission Protease® reaction was dialyzed against 4 L of20 mM Tris-HCl, 10 mM NaCl, pH 8 for 2-4 hours, 4° C. then exchangedwith fresh dialysis buffer and let dialyze overnight. The dialysate wasapplied to 5×5 mL HiTrap Q FF columns equilibrated in 20 mM Tris-HCl,150 mM NaCl, pH 8 (Buffer A), 4° C. The column was washed at 1 mL/minfor 120 min with Buffer A followed by a linear gradient elution of0-100% Buffer B (20 mM Tris-HCl, 1.5 M NaCl, pH 8) over 280 min. TheHip1 containing peak was concentrated to 13 mg/mL and applied to HiPrep16/60 Sephacryl S-100 HR size exclusion column (GE Healthcare) toseparate folded from misfolded and aggregated Hip1. Protein molecularweight and purity was assessed by SDS-PAGE.

Example 2. Cocrystallization of Hip1-Inhibitor Complex and X-RayStructure Solution

Hip1 was concentrated to approximately 10 mg/mL as determined byBradford assay, using an Amicon Ultra-15kDa MWCO spin concentrator(Millipore). To complex Hip1 with the inhibitor (NS-049-2), anapproximate 1:1 molar ratio of inhibitor dissolved in 100% DMSO wasadded to concentrated Hip1 with a final DMSO concentration of 2%. Thecomplex was incubated on ice, overnight, then centrifuged at 14,000 rpm,4° C. to remove precipitated protein. Crystal trials were set up usingthe hanging drop method with 4 ul drops, 2 mL mother liquor at 25° C. Afine screen of HR2-122 #15 (0.17 M ammonium sulfate, 0.085 M Nacacodylate trihydrate, pH 6.5, 25.5% w/v PEG 8,000, 15% v/v glycerol;Hampton Research) was conducted to determine optimal precipitantconditions. Large rods appeared in 5 days in buffer containing 25.5% PEG8000, 0.12 M Ammonium Sulfate, 0.085 M Na Cacodylate trihydrate, pH 6.5,15% v/v glycerol.

Crystallographic data was collected to 2.7 Å on beamline 14—1 at theStanford Synchrotron Radiation Lightsource (SSRL). Data were processedusing HKL2000. The crystal belongs to space group P 3121 and containsone molecule in the asymmetric unit. The structure was solved bymolecular replacement using apoHip1 as a search model (PDB ID 5UNO).Final refinement involved positional, individual b-factor, and TLSrefinement utilizing secondary structure restraints and reference modelrestraints using apoHip 1 (PDB ID SUNO) as a reference model. Phaser-MRand Phenix.refine as implemented in the PHENIX software package was usedfor molecular replacement and refinement, respectively. Model buildingwas performed using Coot. Structure validation was performed usingMolprobity.

Example 3. Minimum Inhibitory Concentration (MIC) Against M.Tuberculosis

MICs were performed as described in Ollinger et al., 2013. Briefly, M.tuberculosis H37Rv expressing DsRed (Caroli etal., 2018) was grown inMiddlebrook 7H9 medium containing 10% OADC (oleic acid, albumin,dextrose, catalase)_supplement (Becton Dickinson) and 0.05% w/v Tween 80(7H9-Tw-OADC) under aerobic conditions. Log phase bacteria wasinoculated in assay plates containing compounds at a highestconcentration of 200 μM using a 10-point two-fold serial dilution in 2%DMSO final concentration. Bacterial growth was measured by OD and RLUafter 5 days of incubation at 37° C. The MIC was defined as the minimumconcentration required for complete inhibition of growth.

Example 4. Intracellular M. Tuberculosis Activity

M. tuberculosis H37Rv constitutively expressing DsRed (Caroll etal.,2018) was grown in Middlebrook 7H9 medium containing 10% v/v OADC (oleicacid, albumin, dextrose, catalase) supplement (Becton Dickinson) and0.05% w/v Tween 80 (7H9-Tw-OADC) at 37° C. under aerobic conditionsuntil log phase. Intracellular M. tuberculosis activity of inhibitorcompounds was determined as described in (Manning et al., 2017).Briefly, RAW 264.7 cells were infected with log phase M. tuberculosisH37Rv constitutively expressing DsRed at a MOI of 1 for 24hr at 37° C.in a humidified 5% CO₂, incubator. Infected cells were washed, harvestedand inoculated in assay plates containing compounds and incubated for 3days at 37° C. in a humidified 5% CO, incubator. Compounds were assayedat a highest concentration of 100 μM using a 10-point three-fold serialdilution in 1% DMSO final concentration. The cellular dye SYBR Green I(10,000X, Thermo Fisher) was added to assay plates at 5× finalconcentration and plates were imaged using an automated ImageXpressMicro XLS High Content Screening System (Molecular Devices) using FITCand Texas Red channels at 4× magnification. Raw data was normalized tonegative control (1% DMSO) and expressed as % growth inhibition.

Example 5. Activity and Low Toxicity in Cellular Assays

Cells were seeded in plates and incubated overnight in a humidifiedincubator at 37° C., 5% CO_(2.) Inhibitor compounds were added 24 hourspost cell seeding to cells at a highest concentration of 100 μM using a10-point three-fold serial dilution in 1% DMSO final concentration.After 72-hours of incubation, CellTiter-Glo® reagent (Promega) was addedto plates and the relative luminescent units (RLU) were measured using aSynergy 4 plate reader (Biotek). Raw data were normalized using theaverage RLU value from negative control (1% DMSO) and expressed as %growth. Growth inhibition curves were fitted using theLevenberg—Marquardt algorithm. The IC₅₀ was defined as the compoundconcentration that produced 50% of the growth inhibitory response.

1. A class of compounds for the inhibition of Hydrolase important forpathogenesis (Hip1), the compounds comprising: A tripeptide targetingsequence, Phe-Lys-Leu, that directs the compound to the active site ofHip1; and A C-terminal alpha-keto methyl ester electrophilic warheadconjugated to the targeting sequence, the warhead configured to inactivethe enzyme.
 2. The compound of claim 1 wherein the electrophilic warheadcomprises (Z)-(X)_(n+1)-(X)-Lys-(X)-(Y), where (Y)=any electrophilicgroup capable of forming a covalent attachment to active site Ser228 ofHip1, where (Z)=any protecting group, (X)_(n+1)=any amino acid(s), aminoacid derivatives, and (X)=any amino acids, amino acid derivatives, orchemistries.
 3. The compound of claim 2 comprising a pharmaceuticalcompound for treatment of tuberculosis comprising the compound.
 4. Thetripeptide targeting sequence of claim 1 comprising at least one of atreatment compound, a diagnostic assay, a molecular probe, and a boostervaccine.
 5. The compound of claim 1 wherein the compound comprises:


6. The compound of claim 5 comprising at least one of a treatmentcompound, a diagnostic assay including from patient sputum, a molecularprobe, and a booster vaccine.
 7. The compound of claim 1 furthercomprising P1 derivatives selected from the group consisting of:Cbz-Phe-Lys-Gln-COCO₂Me; Cbz-Phe-Lys-Gln lactam-COCO₂Me;Cbz-Phe-Lys-Asn-COCO₂Me; Cbz-Phe-Lys-Glu-COCO₂Me;Cbz-Phe-Lys-Val-COCO₂Me; and Cbz-Phe-Lys-(X)-COCO₂Me; wherein (X)comprises any amino acid, amino acid derivative, or chemistry; and thepharmaceutically acceptable salts thereof.
 8. The P1 derivatives ofclaim 7 comprising least one of a treatment compound, a diagnostic assayincluding from patient sputum, a molecular probe for the detection ofserine/threonine proteases involved in pathophysiological processes, anda booster including to a BCG vaccine.
 9. The compound of claim 1 furthercomprising P3 derivatives selected from the group consisting of:Cbz-Tyr-Lys-Leu-COCO₂Me; Cbz-Nle-Lys-Leu-COCO₂Me; andCbz-(X)-Lys-Leu-COCO₂Me; wherein (X) comprises any amino acid, aminoacid derivative, or chemistry; and the pharmaceutically acceptable saltsthereof.
 10. The P3 derivatives of claim 9 comprising at least one of atreatment compound, a diagnostic assay including from patient sputum, amolecular probe for the detection of serine/threonine proteases involvedin pathophysiological processes, and a booster including to a BCGvaccine.
 11. The compound of claim 1 further comprising P1 and P3Derivatives of the compound of claim 1 of the formulaCbz-(X)_(n+1)-Lys-(X)-COCO₂Me; wherein (X_(n+1)) and (X) comprise anyamino acid (s), amino acid derivative (s), or chemistry (ies), and thepharmaceutically acceptable salts thereof.
 12. The P1 and P3 derivativesof claim 11 comprising least one of a treatment compound, a diagnosticassay including from patient sputum, a molecular probe for the detectionof serine/threonine proteases involved in pathophysiological processes,and a booster including to the BCG vaccine.
 13. The compound of claim 1further comprising Truncated derivatives selected from the groupconsisting of: Cbz-Leu-COCO₂Me; Cbz-X-COCO₂Me, wherein (X) comprises anyamino acid, amino acid derivative, or chemistry; Cbz-Lys-Leu-COCO₂Me;and Cbz-Lys-X-COCO₂Me; wherein (X) comprises any amino acid, amino acidderivative, or chemistry; and the pharmaceutically acceptable saltsthereof.
 14. The truncated derivatives of claim 11 comprising at leastone of a treatment compound a diagnostic assay including from patientsputum, a molecular probe for the detection of serine/threonineproteases involved in pathophysiological processes, and a boostervaccine.
 15. The compound of claim 1 further comprising N-Terminallengthened derivatives of the formula Cbz-(X)_(n+1)-Phe-Lys-Leu-COCO₂Meor the pharmaceutically acceptable salt thereof wherein (X)_(n+1)comprises any amino acid(s), amino acid derivatives, or chemistries. 16.The N-Terminal lengthened derivatives of claim 15 comprising at leastone of a treatment compound, a diagnostic assay including from patientsputum, a molecular probe for the detection of serine/threonineproteases involved in pathophysiological processes, and a boosterincluding to the BCG vaccine.
 17. The compound of claim 1 furthercomprising protecting group derivatives of the formula(Z)-(X)_(n+1)-(X)-Lys-(X)-COCO₂Me or the pharmaceutically acceptablesalts thereof wherein (Z) comprises any protecting group, (X)_(n+1)comprises any amino acid(s), amino acid derivatives, (X) comprises anyamino acids, amino acid derivatives, or chemistries.
 18. The protectinggroup derivatives of claim 17 comprising at least one of a treatmentcompond, a diagnostic assay including from patient sputum, a molecularprobe for the detection of serine/threonine proteases involved inpathophysiological processes, and a booster including to the BCGvaccine.
 19. The compound Cbz-Phe-Lys-Leu-pNa (pNa=paranitroanilide) orderivatives thereof including conjugation to various chromophores,fluorophores, antibodies, nanobodies, or other reporter groups for thedesign of novel enzymatic activity assays, serine protease purificationmethods, molecular probes for the detection of novel serine/threonineproteases, and rapid diagnostic tests for the presence of Mtb inpatients.
 20. The compound of claim 19 further comprising at least oneof a treatment compound, a diagnostic assay including from patientsputum, a molecular probe for the detection of serine/threonineproteases involved in pathophysiological processes, and a boosterincluding to the BCG vaccine.